The present invention relates to methods and pharmaceutical compositions for inducing and/or accelerating cell proliferation and/or cell migration and/or cell differentiation and thereby accelerating the healing process of wounds. More particularly, the present invention relates to the use of bioactive molecules which are secreted by adipocytes (adipokines), and bioactive molecules which modulate adipocytes and preadipocytes differentiation, and/or adipocytes proliferation and/or activity and/or chemotaxis, for inducing or accelerating the healing process of a damaged skin or skin wound.
The primary goal in the treatment of wounds is to achieve wound closure. Open cutaneous wounds represent one major category of wounds and include surgical wounds, burn wounds, neuropathic ulcers, pressure sores, venous stasis ulcers, and diabetic ulcers.
Open cutaneous wounds routinely heal by a process that comprises six major components: (i) inflammation; (ii) fibroblast proliferation; (iii) blood vessel proliferation; (iv) connective tissue synthesis; (v) epithelialization; and (vi) wound contraction. Wound healing is impaired when these components, either individually or as a whole, do not function properly. Numerous factors can affect wound healing, including malnutrition, infection, pharmacological agents (e.g., actinomycin and steroids), advanced age and diabetes (Hunt and Goodson, 1988).
With respect to diabetes, diabetes mellitus is characterized by impaired insulin signaling, elevated plasma glucose and a predisposition to develop chronic complications involving several distinctive tissues. Among all the chronic complications of diabetes mellitus, impaired wound healing leading to foot ulceration is among the least well studied. Yet skin ulceration in diabetic patients takes a staggering personal and financial cost (Knighton and Fiegel, 1993; Shaw and Boulton, 1997). Moreover, foot ulcers and the subsequent amputation of a lower extremity are the most common causes of hospitalization among diabetic patients (Shaw and Boulton, 1997; Coghlan et al., 1994; Grunfeld, 1992; Reiber et al., 1998). In diabetes, the physiological process of wound healing is impaired. The defect in tissue repair has been related to several factors including neuropathy, vascular disease and infection. However, other mechanisms whereby the diabetic state associated with abnormal insulin signaling impairs wound healing and alters the physiology of skin has not been elucidated.
Another issue associated with impaired wound healing relates to infections of post surgical wounds occurring in 25% of patients hospitalized in surgical wards.
Skin is a stratified squamous epithelium in which cells undergoing growth and differentiation are strictly compartmentalized. In the physiologic state, proliferation is confined to the basal cells that adhere to the basement membrane. Differentiation is a spatial process where basal cells lose their adhesion to the basement membrane, cease DNA synthesis and undergo a series of morphological and biochemical changes. The ultimate maturation step is the production of the cornified layer forming the protective barrier of the skin (Hennings et al., 1980; Yuspa et al., 1989). The earliest changes observed when basal cells commit to differentiate is associated with the ability of the basal cells to detach and migrate away from the basement membrane (Fuchs, 1990). Similar changes are associated with the wound healing process where cells both migrate into the wound area and proliferative capacity is enhanced. These processes are mandatory for the restructuring of the skin layers and induction of proper differentiation of the epidermal layers.
The analysis of mechanisms regulating growth, differentiation and migration of epidermal cells has been greatly facilitated by the development of culture systems for mouse and human keratinocytes (Yuspa et al., 1989; Yuspa, 1994). In vitro, keratinocytes can be maintained as basal proliferating cells with a high growth rate. Furthermore, differentiation can be induced in vitro following the maturation pattern in the epidermis in vivo. The early events include loss of hemidesmosome components (Fuchs, 1990, Hennings and Holbrook, 1983) and a selective loss of the α6β4 integrin and cell attachment to matrix proteins. This suggests that changes in integrin expression are early events in keratinocyte differentiation. The early loss of hemidesmosomal contact leads to suprabasal migration of keratinocytes and is linked to induction of Keratin 1 (K1) in cultured keratinocytes and in skin (Hennings et al., 1980; Fuchs, 1990; Tennenbaum et al., 1996a). Further differentiation to the granular layer phenotype is associated with down regulation of both β1 and β4 integrin expression, loss of adhesion potential to all matrix proteins and is followed by cornified envelope formation and cell death. Differentiating cells ultimately sloughs from the culture dish as mature squames (Yuspa et al., 1989; Tennenbaum et al., 1996b). This program of differentiation in vitro closely follows the maturation pattern of epidermis in vivo.
Wound healing may be induced in vivo by various bioactive agents which directly or indirectly promote growth, differentiation and/or migration of keratinocytes and/or epidermal cells. Thus, U.S. Pat. Nos. 5,591,709 and 5,461,030 describe the use of non-steroidal anabolic hormone such as insulin, growth hormone, triiodothyronine and thyroxine for inducing wound closure. U.S. Pat. No. 5,145,679 describes the use of insulin and pancreatin for inducing wound closure. U.S. Pat. No. 6,541,447 describes the use of a mixture of growth factors and growth hormones for inducing wound closure, and International Application No. PCT/IL01/00675 (WO 02/09639) and corresponding U.S. patent application Ser. No. 09/169,801 describe the use of PKC modulating agents for inducing wound closure. However, there is no teaching in the prior art for utilizing adipocytes, adipocyte or preadipocyte modulators, or molecules secreted by adipocytes, for inducing or accelerating the processes associated with wound healing.
There is thus a widely recognized need for, and it would be highly advantageous to have, new approaches for accelerating the processes associated with wound healing.